The Helicobacter pylori HrcA repressor: Study of the Global Transcriptional Response during Heat Shock.

Ojaimi Loibman, Stefany (2019) The Helicobacter pylori HrcA repressor: Study of the Global Transcriptional Response during Heat Shock., [Dissertation thesis], Alma Mater Studiorum Università di Bologna. Dottorato di ricerca in Biologia cellulare e molecolare, 31 Ciclo. DOI 10.6092/unibo/amsdottorato/8964.
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The ability to respond quickly to environmental changes and thus modulate gene expression is a crucial skill exploited by bacteria for their survival. In this context, the widespread human pathogen Helicobacter pylori, under stress conditions induces the synthesis of a class of highly conserved proteins, called Heat Shock Proteins. In this bacterium, the major heat-shock genes are negatively regulated by two transcriptional repressors, HspR and HrcA. Although the heat-shock regulatory circuit is well-studied, not enough is known about the global heat shock response in H. pylori. In order to identify other potential cellular process regulated by HrcA, we performed differential transcriptional analysis of heat shock regulation by RNA-seq, comparing the transcriptome of the H. pylori G27 wild type strain subjected and not subjected to heat shock stress versus the HrcA isogenic G27 mutant strain. This analysis showed that most of the differentially expressed genes were deregulated only under heat shock treatment. Moreover, several non-heat shock responsive genes were deregulated in the HrcA mutant. To further characterize the HrcA regulon in H. pylori we attempted setting up a ChIP-seq experiment. However, the HrcA protein appears to be poorly immunogenic, for this reason we used a strategy in which an epitope (3XFLAG), recognized by commercial antibodies, was fused to the N-terminal region of the HrcA protein. In addition, three levels of expression (weak, intermediate and strong) of the 3XFLA-HrcA were obtained by using three known H. pylori promoters. The immunoprecipitation assays performed showed a poor enrichment of DNA fragments bound by HrcA and hindered the identification of novel in vivo HrcA binding sites. Thereafter, we further analyzed the HrcA-DNA interactions on some putative targets through DNaseI footprinting assays on novel putative target promoters binding of the regulator could not be detected and possibly transcriptional regulation by HrcA of these genes is indirect.

Tipologia del documento
Tesi di dottorato
Ojaimi Loibman, Stefany
Dottorato di ricerca
Settore disciplinare
Settore concorsuale
Parole chiave
Helicobacter pylori Heat-shock Response HrcA
Data di discussione
2 Aprile 2019

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